Direct Interaction of a Divergent CaM Isoform and the Transcription Factor, MYB2, Enhances Salt Tolerance in Arabidopsis

Calmodulin (CaM), a ubiquitous calcium-binding protein, regulates diverse cellular functions by modulating the activity of a variety of enzymes and proteins. Plants express numerous CaM isoforms that exhibit differential activation and/or inhibition of CaM-dependent enzymes in vitro. However, the specific biological functions of plant CaM are not well known. In this study, we isolated a cDNA encoding a CaM binding transcription factor, MYB2, that regulates the expression of salt- and dehydration-responsive genes in Arabidopsis. This was achieved using a salt-inducible CaM isoform (GmCaM4) as a probe from a salt-treated Arabidopsis expression library. Using domain mapping, we identified a Ca2+-dependent CaM binding domain in MYB2. The specific binding of CaM to CaM binding domain was confirmed by site-directed mutagenesis, a gel mobility shift assay, split ubiquitin assay, and a competition assay using a Ca2+/CaM-dependent enzyme. Interestingly, the specific CaM isoform GmCaM4 enhances the DNA binding activity of AtMYB2, whereas this was inhibited by a closely related CaM isoform (GmCaM1). Overexpression of Gm-CaM4 in Arabidopsis up-regulates the transcription rate of AtMYB2-regulated genes, including the proline-synthesizing enzyme P5CS1 (Δ1-pyrroline-5-carboxylate synthetase-1), which confers salt tolerance by facilitating proline accumulation. Therefore, we suggest that a specific CaM isoform mediates salt-induced Ca2+ signaling through the activation of an MYB transcriptional activator, thereby resulting in salt tolerance in plants. Calmodulin (CaM), a ubiquitous calcium-binding protein, regulates diverse cellular functions by modulating the activity of a variety of enzymes and proteins. Plants express numerous CaM isoforms that exhibit differential activation and/or inhibition of CaM-dependent enzymes in vitro. However, the specific biological functions of plant CaM are not well known. In this study, we isolated a cDNA encoding a CaM binding transcription factor, MYB2, that regulates the expression of salt- and dehydration-responsive genes in Arabidopsis. This was achieved using a salt-inducible CaM isoform (GmCaM4) as a probe from a salt-treated Arabidopsis expression library. Using domain mapping, we identified a Ca2+-dependent CaM binding domain in MYB2. The specific binding of CaM to CaM binding domain was confirmed by site-directed mutagenesis, a gel mobility shift assay, split ubiquitin assay, and a competition assay using a Ca2+/CaM-dependent enzyme. Interestingly, the specific CaM isoform GmCaM4 enhances the DNA binding activity of AtMYB2, whereas this was inhibited by a closely related CaM isoform (GmCaM1). Overexpression of Gm-CaM4 in Arabidopsis up-regulates the transcription rate of AtMYB2-regulated genes, including the proline-synthesizing enzyme P5CS1 (Δ1-pyrroline-5-carboxylate synthetase-1), which confers salt tolerance by facilitating proline accumulation. Therefore, we suggest that a specific CaM isoform mediates salt-induced Ca2+ signaling through the activation of an MYB transcriptional activator, thereby resulting in salt tolerance in plants. In both plants and animals, Ca2+ mediates stimulus-response coupling in the regulation of diverse cellular functions that are triggered by a variety of biotic and abiotic external stimuli (1Knight M.R. Campbell A.K. Smith S.M. Trewavas A.J. Nature. 1991; 352: 524-526Crossref PubMed Scopus (932) Google Scholar, 2Bush D.S. Annu. Rev. Plant Mol. Biol. 1995; 46: 95-122Crossref Scopus (915) Google Scholar, 3Price A.H. Taylor A. Ripley S.J. Griffiths A. Trewavas A.J. Knight M.R. 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Calmodulin (CaM), 1The abbreviations used are: CaM, calmodulin; CaMBP, CaM-binding proteins; HRP, horseradish peroxidase; IPTG, isopropyl 1-thio-β-d-galactopyranoside; TBS, Tris borate-EDTA; GST, glutathione S-transferase; PDE, phosphodiesterase; EMSA, electrophoretic mobility shift assay; GUS, β-glucuronidase; Ub, ubiquitin; Nub, N-terminal half of ubiquitin; Cub, C-terminal half of ubiquitin; RUra3p, Ura3p reporter containing Arg in amino acid position 1; 5-FOA, 5-fluoroorotic acid. a ubiquitous Ca2+-binding protein with four EF hands, is a well characterized Ca2+ sensor that is known to modulate the activity of many mammalian proteins. The molecular targets of plant CaM are under active investigation (7Lu K.P. Means A.R. Endocr. Rev. 1993; 14: 40-58Crossref PubMed Scopus (272) Google Scholar, 8Crivici A. Ikura M. Annu. Rev. Biophys. Biomol. Struct. 1995; 24: 85-116Crossref PubMed Scopus (697) Google Scholar). Various plant species have been shown to possess multiple CaM genes with as many as 20 CaM isoforms present in Arabidopsis (9Initiative The Arabidopsis Genome Nature. 2000; 408: 796-815Crossref PubMed Scopus (7211) Google Scholar). The induction of individual CaM genes is differentially regulated in response to physical (e.g. touch, heat, and light) and chemical (e.g. auxin and fungal elicitor) stimuli (10Botella J.R. Arteca R.N. Plant Mol. Biol. 1994; 24: 757-766Crossref PubMed Scopus (66) Google Scholar, 11Heo W.D. Lee S.H. Kim M.C. Kim J.C. Chung W.S. Chun H.J. Lee K.J. Park C.Y. Park H.C. Choi J.Y. Cho M.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 766-771Crossref PubMed Scopus (216) Google Scholar). In turn, these CaM isoforms differentially regulate 20 Ca2+/CaM-dependent enzymes, suggesting that CaM isoforms sense different Ca2+ signals and transduce the signals to diverse sets of CaM-binding proteins (CaMBPs), thereby generating physiological responses to stimuli (12Lee S.H. Kim M.C. Heo W.D. Kim J.C. Chung W.S. Park C.Y. Park H.C. Cheong Y.H. Kim C.Y. Lee K.J. Bahk J.D. Lee S.Y. Cho M.J. Biochim. Biophys. Acta. 1999; 1433: 56-67Crossref PubMed Scopus (44) Google Scholar, 13Liao B. Paschal B.M. Luby-Phelps K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6217-6222Crossref PubMed Scopus (49) Google Scholar, 14Zuhlke R.D. Pitt G.S. Deisseroth K. Tsien R.W. Reuter H. Nature. 1999; 399: 159-162Crossref PubMed Scopus (744) Google Scholar). In addition to regulating enzyme activity, Ca2+-loaded CaM modulates transcriptional activators. Some transcription factors are modulated indirectly through phosphorylation by Ca2+/CaM-dependent protein kinases (15Corcoran E.E. Means A.R. J. Biol. Chem. 2001; 276: 2975-2978Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), whereas basic helix-loop-helix groups interact directly with Ca2+/CaM, resulting in the DNA binding activity of basic helix-loop-helix being inhibited (16Corneliussen B. Holm M. Waltersson Y. Onions J. Hallberg B. Thornell A. Grundstrom T. Nature. 1994; 368: 760-764Crossref PubMed Scopus (144) Google Scholar). MYB proteins constitute a diverse class of DNA-binding proteins and are particularly important for transcriptional regulation in plants. This protein family is characterized by having a structurally conserved DNA binding domain, the MYB domain, and is classified into three subfamilies on the basis of the number of imperfect adjacent repeats of the MYB DNA binding domain. The two-repeat (R2R3) MYB family is the most commonly of those identified in plants and is estimated to consist of 125 members in Arabidopsis (17Stracke R. Werber M. Weisshaar B. Curr. Opin. Plant Biol. 2001; 4: 447-456Crossref PubMed Scopus (1534) Google Scholar). The known functions of plant MYB proteins include the regulation of secondary metabolism, control of cellular morphogenesis, and regulation of the meristem and the cell cycle (18Kranz H.D. Denekamp M. Greco R. Jin H. Leyva A. Meissner R.C. Petroni K. Urzainqui A. Bevan M. Martin C. Smeekens S. Tonelli C. Paz-Ares J. Weisshaar B. Plant J. 1998; 16: 263-276Crossref PubMed Google Scholar). Although some functional similarities exist among R2R3 MYB proteins that are closely related structurally, significant functional differences exist both in the and in different species (18Kranz H.D. Denekamp M. Greco R. Jin H. Leyva A. Meissner R.C. Petroni K. Urzainqui A. Bevan M. Martin C. Smeekens S. Tonelli C. Paz-Ares J. Weisshaar B. Plant J. 1998; 16: 263-276Crossref PubMed Google Scholar). In this study, we from an Arabidopsis cDNA expression using horseradish CaM as a probe and that CaM with AtMYB2, a transcription that regulates salt- and dehydration-responsive in in and in that CaM isoforms differentially regulate the DNA binding activity of GmCaM4 in Arabidopsis the transcription of AtMYB2-regulated genes including that of the proline-synthesizing which confers salt tolerance to these plants by to proline that Ca2+ signaling by a specific CaM isoform to salt tolerance in plants. of the Arabidopsis cDNA Arabidopsis cDNA expression was in a from Arabidopsis with for CaM isoforms to a using the as in a (12Lee S.H. Kim M.C. Heo W.D. Kim J.C. Chung W.S. Park C.Y. Park H.C. Cheong Y.H. Kim C.Y. Lee K.J. Bahk J.D. Lee S.Y. Cho M.J. Biochim. Biophys. Acta. 1999; 1433: 56-67Crossref PubMed Scopus (44) Google Scholar). the Arabidopsis cDNA expression using GmCaM4 as a using as the The and with that been in The was for which to The and in a of containing The by in and the three with for and in for The a by in containing and for was to the a concentration of and the for was in three with of repeats of a in and in 20 and and in 20 and was using an of and isolated three of cDNA by in with the binding of to CaM, we as proteins in and the for CaM binding using a assay as into and the expression of proteins with 20 of protein on a gel and an The was in by in and as of binding of CaM, was for in of the The cDNA of the resulting from both of cDNA by using an DNA of cDNA and of with the amino and and and and and and was to the of into a the in the CaM and AtMYB2, we into the of amino using the site-directed The and used are as for and and for and of in and CaM of the into and The expression of the proteins was by of in and on for 20 The was for using a and for to cell proteins on a of protein was on and which proteins using a the CaM binding of a was with a in the of The assay was as The CaM was using an CaM with a to a of amino in the of was S. The CaM binding of the was by the mobility shift of CaM in the of this S. PubMed Scopus Google Scholar). CaM was with of the and in a binding for of the was and the in containing and The a of in an and and with using The with of GmCaM4 and a concentration of of the and a concentration of GmCaM4 The was with the addition of enzyme activity was in the of and activity was in the of GmCaM4 and for the was by the into a for and on for a of was and for The was by of acid. the and the was to a of S.H. Kim J.C. Lee Heo W.D. J.C. Lee S.Y. Bahk J.D. Cho M.J. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), the was for and for The of for the was from the concentration of to activity in the of The was used to S. PubMed Scopus Google is the concentration of and and are the of CaM to activation of in the of gel was as by Lee S.H. Kim J.C. Lee Heo W.D. J.C. Lee S.Y. Bahk J.D. Cho M.J. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). split ubiquitin assay was as H. C. J. S. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). was used for of the and Gm-CaM4 into the cDNA was into of the C. S. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). The of of proteins was on a containing and on a for for was as a and a DNA probe was by in the with and a The DNA binding was to for 20 in binding that of DNA of and of protein that been with of from plants in the of The was to on an gel in for The gel was and for with reporter was a containing the reporter under the control of a T. S. J.D. C. H. R. 1995; PubMed Scopus Google Scholar). was in of the The was to a to the reporter The of the Arabidopsis P5CS1 was by Y. T. S. K. K. Biophys. 1999; PubMed Scopus Google and to a to the reporter of AtMYB2, GmCaM4 cDNA into a plant expression containing the and J. PubMed Scopus Google Scholar). expression of these was as A. A.K. Plant Mol. Biol. 1997; PubMed Scopus Google Scholar). In with of DNA a reporter with DNA an a DNA The for in the the to the was used as an control in in which the reporter was In the activity of the cell was by the activity, thereby the to control for in the Plant cell was in as Plant Google Scholar). that been for was used for the was with for the of with A. was The plants on a containing basic and Plant and of GmCaM4 into a plant in which the isoform under the control of the in the sense The into and Arabidopsis plants by a of plants of GmCaM4 used for of the the salt tolerance on to with with the in the the concentration of was by and for plants in and in plant was in acid to amino amino with an amino acid of a cDNA a CaM-binding with Plant to salt J. Knight M.R. Plant J. 2000; PubMed Google Scholar). specific Ca2+-binding protein with a Ca2+ binding domain been shown to a of salt for salt in plant J. 1998; PubMed Scopus Google Scholar). we that the transcription of specific CaM isoforms are by a W.D. Lee S.H. Kim M.C. Kim J.C. Chung W.S. Chun H.J. Lee K.J. Park C.Y. Park H.C. Choi J.Y. Cho M.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 766-771Crossref PubMed Scopus (216) Google Scholar). the expression of CaM genes is in response to abiotic that we cell with salt and a isolated from cell was and and for The of GmCaM4 was by not by the In a expression was not by that a specific CaM isoform in salt signaling in plants. with salt a cDNA from Arabidopsis was using GmCaM4 this CaM (12Lee S.H. Kim M.C. Heo W.D. Kim J.C. Chung W.S. Park C.Y. Park H.C. Cheong Y.H. Kim C.Y. Lee K.J. Bahk J.D. Lee S.Y. Cho M.J. Biochim. Biophys. 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Plant Cell. 2000; Google the Calmodulin of of the of many shown that are multiple for CaM A.R. J. 1997; PubMed Scopus Google Scholar). on the of known a CaM binding was in the R2R3 DNA binding of and and this acid amino present and and basic among these of the a of basic and on with amino on the the of AtMYB2, we a of containing the cDNA and four The proteins in by and to a for for a of the protein was by the with an proteins that the and with both the and whereas and the proteins the and not interact with CaM that the and CaM is we the binding of CaM to MYB protein, K. K. T. M. Plant Mol. Biol. PubMed Scopus Google Scholar), which CaM binding shown in CaM isoforms not to a In we the binding of CaM to of which a CaM binding number and number in (18Kranz H.D. Denekamp M. Greco R. Jin H. Leyva A. Meissner R.C. Petroni K. Urzainqui A. Bevan M. Martin C. Smeekens S. Tonelli C. Paz-Ares J. Weisshaar B. Plant J. 1998; 16: 263-276Crossref PubMed Google with In CaM-binding protein protein was to a CaM-binding isoforms to the of in the not in the of Ca2+ the of amino acid in CaM we the and in the with Arg by and and the for CaM binding in a CaM shown in not to the amino acid amino acid whereas GmCaM4 to the amino acid not to the amino acid that CaM with in a Ca2+-dependent and that these are for the of a to the binding of CaM to the acid of from to a to this was and used for a gel shift assay in which the CaM to the CaM under S. PubMed Scopus Google Scholar). shown in the of the molecular the with concentration of the in the of whereas the molecular was was for a of CaM was whereas of the CaM was of and the binding of the to by competition assay PDE, a CaM-dependent enzyme. the of the for activation of by the activation of was in the of the the concentration of the was activity with of CaM resulting in a shift of the activation in the of the in activity was of the and a concentration of CaM used The of to activation of activity in the and of the and a The of the for activation of by was to The of Gm-CaM4 to activity and in the and of the a The of the for the activation of by GmCaM4 was to that the binding of GmCaM4 for the is that of of in CaM and with a in the the protein and we used a split ubiquitin assay which is on the of the and C-terminal and of ubiquitin protein and isoforms to the of and the of Cub, The of ubiquitin was to an Ura3p reporter containing Arg in amino acid position In this assay, the isoforms interact with the protein, and into a and by The through the of protein A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). the containing and to on to on containing this into by the not by the the the and and to on to on containing 5-FOA, that the protein both with and GmCaM4 in was and the and the of the the with containing amino acid in the in protein proteins on not and a of GmCaM4 and the a to whereas the of GmCaM4 and the not on the as with CaM the DNA of the in differences in the of individual isoforms to the transcriptional suggest that CaM isoforms differentially regulate the DNA binding activity of and the control of AtMYB2-regulated we have shown that and whereas GmCaM4 as a of activation S.H. Kim J.C. Heo W.D. Chung W.S. Lee K.J. Kim M.C. Cheong Y.H. Choi J.Y. Cho M.J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The is for mammalian M.J. R. Lee S.H. R. Heo W.D. J.D. 1998; PubMed Scopus Google Scholar). Therefore, we and GmCaM4 differentially regulate the DNA binding activity of the T. K. S. K. Plant Cell. 1993; PubMed Scopus Google Scholar, K. K. Plant 1997; PubMed Scopus Google using Interestingly, the DNA binding activity of was in a Ca2+-dependent by with whereas this activity was inhibited by and The differential regulation of DNA binding activity by GmCaM4 and was the of the individual CaM isoforms with a CaM concentration of The DNA binding activity of the containing the amino acid was not by was that of the by GmCaM4 are with the CaM binding of of CaM and in Arabidopsis isoforms directly to the transcription and differentially regulate DNA binding of the to in we the isoform proteins regulate transcription of salt- and dehydration-responsive genes using the Arabidopsis with a reporter to the of the DNA containing the T. S. J.D. C. H. R. 1995; PubMed Scopus Google Scholar). The of the to AtMYB2, GmCaM4 shown in the expression of GmCaM4 in the the expression of the reporter and with the a induction of reporter expression was in in which GmCaM4 and In expression was inhibited in and with expression was not by the of GmCaM4 with of for and for that are of binding to CaM isoforms the CaM isoforms the expression of salt- and dehydration-responsive genes, the to from the transcription of an Arabidopsis proline was by Y. T. S. K. K. Biophys. 1999; PubMed Scopus Google Scholar). The reporter was to the and transient with and was as The reporter was by GmCaM4 by and However, the expression of the reporter by the P5CS1 was by with Gm-CaM4 and of reporter expression by was not in this Overexpression of a CaM (GmCaM4) in the of and GmCaM4 on expression in we Arabidopsis plants GmCaM4 under the control of the and expression of salt- and dehydration-responsive genes, proline and salt P5CS1 is a enzyme in the plant proline the of GmCaM4 in Arabidopsis plants the expression of salt- and dehydration-responsive genes as and and the proline concentration of the GmCaM4 plants was with that of plants the GmCaM4 Arabidopsis plants the in the of is not this of the in Gm-CaM4 plants is by regulation the regulation of by this we an using protein This that proteins from GmCaM4 plants to the DNA binding proteins from plants plants this binding was by the addition of the DNA probe salt tolerance in and plants isoforms on containing of the as well as plants well and on control the not containing the of control and plants was whereas the of GmCaM4 plants was not inhibited and In and plants and a inhibition with with the plants GmCaM4 salt tolerance in response to with plants The three a of GmCaM4 and salt tolerance the DNA binding activity of was inhibited by with in and in and proline and salt tolerance not in plants. The molecular of plant salt tolerance are and are not well are known to as a in salt signaling in plants H. Trewavas A.J. Knight M.R. Plant J. 1997; PubMed Scopus Google Scholar). that CaM as a Ca2+ sensor of a variety of external we that CaM isoforms differentially regulate the activation of 20 Ca2+/CaM-dependent enzymes, that CaM isoforms sense different Ca2+ signals by external stimuli and transduce these signals to a diverse of S.H. Kim J.C. Heo W.D. Chung W.S. Lee K.J. Kim M.C. Cheong Y.H. Choi J.Y. Cho M.J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus (66) Google Scholar, M.J. R. Lee S.H. R. Heo W.D. J.D. 1998; PubMed Scopus Google Scholar, S.H. J.D. C. A. H. Cho M.J. J. 2000; PubMed Scopus Google Scholar). with salt we a Arabidopsis cDNA expression library. a we isolated a CaM-binding protein, AtMYB2, which is an MYB transcriptional are known to regulate expression of salt- and dehydration-responsive genes as P5CS1 Y. T. S. K. K. Biophys. 1999; PubMed Scopus Google Scholar), R. Y. 1998; PubMed Google Scholar), and H. K. T. T. K. Plant Cell. 1997; Google Scholar). The CaM binding of was a of amino and which the R2R3 DNA binding of This a The of a amino Arg to the conserved in the the and The of amino Arg and Arg to the and in the the GmCaM4 and the binding of GmCaM4 for is to that of for The differential binding of the isoforms for the transcriptional suggest that CaM isoforms differentially regulate DNA binding activity and thereby was that and whereas GmCaM4 as a of these S.H. Kim J.C. Heo W.D. Chung W.S. Lee K.J. Kim M.C. Cheong Y.H. Choi J.Y. Cho M.J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The is for the regulation of mammalian which is by Gm-CaM4 and inhibited by M.J. R. Lee S.H. R. Heo W.D. J.D. 1998; PubMed Scopus Google Scholar). in mammalian with CaM and protein have shown that CaM isoforms have different for proteins Means A.R. H. J. 1994; PubMed Scopus Google Scholar). induction of reporter expression was by GmCaM4 and In reporter expression was not inhibited by the expression of and with the expression of However, in transient using the P5CS1 not DNA binding in Arabidopsis the expression of AtMYB2-regulated genes and proline was suggest that factors in addition to in P5CS1 CaM been to to the DNA binding and of basic helix-loop-helix transcriptional thereby DNA binding to (16Corneliussen B. Holm M. Waltersson Y. Onions J. Hallberg B. Thornell A. Grundstrom T. Nature. 1994; 368: 760-764Crossref PubMed Scopus (144) Google Scholar). In CaM to the transcription and enhances binding to the B. R.E. Plant Cell. PubMed Scopus Google Scholar). However, differential regulation by CaM isoforms of the DNA binding activity of transcriptional not been in plants to the binding of individual isoforms to the R2R3 DNA binding domain of differentially regulates the DNA binding activity of this transcription Although from on CaM from particularly important in the of CaM isoforms in plants. a with a protein of from suggesting the of a GmCaM4 in Arabidopsis S.H. Kim J.C. Lee Heo W.D. J.C. Lee S.Y. Bahk J.D. Cho M.J. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). However, to a functional GmCaM4 in Arabidopsis have been plants as proline Curr. Opin. Plant Biol. 1999; PubMed Scopus Google and a to to salt G.S. 1999; PubMed Scopus Google Scholar). The in Arabidopsis is modulated by a protein which is by a Ca2+-binding protein S.H. J.D. C. A. H. Cho M.J. J. 2000; PubMed Scopus Google Scholar). In this we that a specific isoform is in plant salt is to that plants different Ca2+ to salt signals through different The these to characterized this that a specific CaM isoform confers salt tolerance in Arabidopsis through the of activity, resulting in the of salt- and dehydration-responsive genes and the of S. for and on the K. for the and R. for the and R. for the of and the for the split ubiquitin and M. for of the